The development of microfluidic devices, or so called lab-on-a-chip, enabled the miniaturization of large-scale laboratory systems to the size of a chip and has several advantages over conventional methods such as portability, reduction in the amount of reagents and sample, easy implementation of high throughput methods and its small size. Microfluidic devices have various applications in biotechnology and pharmaceutical industry from oxygenation devices, sequencing chips, analytic devices, micro-fuel cells to “plant on a chip”, which are microfluidic chips developed for stimulating for example plant roots with various chemicals. WO2010/018499 discloses microfluidic devices for cellular susceptibility testing via a concentration gradient, and others such as CN201628717 disclose a microfluidic chip for detecting pathogenic bacteria by bioluminescence based on the recombinant firefly luciferase detection system.
Bioluminescence is frequently used in reporter assays, monitoring tumour growth and in vivo imaging (10, 11) and is based on an enzymatic reaction which results in the detectable emission of light. The reaction is based on the conversion of the substrate luciferin to oxyluceferin by the luciferase enzyme which results in emission of light. This oxidative reaction is usually, ATP dependent as in firefly luciferase. Unlike fluorescence, bioluminescence does not require an external light source to excite the chemical reaction thus avoiding photo-bleaching effects. In addition, the virtually zero background allows one to detect bioluminescent signals with high sensitivity. This property of bioluminescence makes it a robust method for high throughput cell screening assays (12-14).
Other luciferase enzymes such as the Gaussia luciferase (GLuc) (9), derived from the marine copepods, brighter when compared to Firefly or Renilla Luciferases (15) and, most importantly, the light emitting reaction is ATP independent. Thus artefacts commonly caused by cellular ATP during signal generation have no effect on the signal generated.
Cell surface receptors play an important role in signal communication and response. The development of a diseased state can also be due to changes in receptor density through up-regulation or down-regulation and a disturbed balance in the (in) activation of the receptors (1). For instance, the change in dopamine D2 receptors in Parkinson's disease and myocardial β1 adrenoceptor down regulation in heart failure are good examples to portray the effect of receptor number on diseases (2, 3).
Thus, there is a need to quantify the number of receptors expressed on both healthy and diseased cells.
There are several approaches to quantify receptor expression in cells. Methods such as the saturation and competitive binding techniques are often used to determine receptor numbers in live or fixed cells (4). The use of radio-labels in the case of saturation and competition binding methods is problematic. Recent developments in near-field scanning optical microscopy (NSOM) techniques using fluorescent probes have also enabled a deterministic method of distribution and quantification of cell surface receptors (5, 6). A localized evanescent wave produced at the tip of the NSOM probe helps to excite the receptor bound fluorescent-ligand to get an image of a target receptor at the nanometer scale resolution. Although the near field imaging technique is a very good method for quantifying cell surface receptors, it works best on fixed cells where the probe to sample distance can be controlled more accurately using a force-feedback loop. In addition, photo-bleaching of the fluorophores can also commonly occur on dry samples due to direct contact with air (7, 8).
This disclosure relates to an assay device, such as a microfluidic device, based bioluminescence and its use in a method to quantify cell surface ligands/receptors. In addition the method can also quantify intracellular receptors by permeabilizing the cell membrane. The device provides an improved, highly sensitive and specific dynamic system for the growth of cells and detection of cell surface molecules on living cells. In addition, without the need for an external illumination, the size of the equipment can be reduced, and as signals detected are directly related to the concentration of the molecule being studied. The approach allows real time signal detection with fast response times.